Free radical and reactive oxygen species (ROS) play an important in the etiology and/or progression of a number of diseases and in aging as well as in signal transduction. Investigators in the Section on Metabolic Regulation carried out studies to elucidate mechanisms by which free radicals and ROS are generated and exert their biological effects. During this fiscal year, we continued to focus on the roles of glutathionylation of actin and protein phosphatase 1B induced by ROS on cell signaling. This investigation involved the use of RNAi. In order to use this method for our specific requirements, a new method was developed and a mechanistic study of this method is being carried out. In addition, hydrogen peroxide was used to investigate the response of B cells to oxidative stress with respect to calcium mobilization mediated by tyrosine phosphatase CD45 and activation of the PI3K/Akt pathway. Previously, we showed that both millimolar concentrations of manganese (II) and serum deprivation led to apoptosis in NIH3T3 cells and neuroblastoma SH-SY5Y cells, respectively. In both cases, elevation of ROS levels was observed. Using RNAi, in the case of the manganese-induced system, we identified that pro-caspase-12, activated by calpain, is responsible for initiating the apoptosis. In the case of serum deprivation-induced apoptosis, we observed the hormesis effect and this anti-apoptotic effect is mediated by a cyclic GMP-dependent protein kinase pathway, initiated by nitric oxide synthesis, that involves expression of thioredoxin and thioredoxin peroxidase-1.